A. Field of the Invention
The present invention relates generally to the field of molecular biology. More particularly, certain embodiments concern the transfer of genetic material into central nervous system cells. In certain examples, the invention concerns the use of liposome-mediated delivery of nucleic acids encoding neurotrophic factors to stimulate nervous system cell repair and regeneration. Rescue of neurofilament protein following traumatic brain injury (TBI) in vivo has been demonstrated.
B. Description of the Related Art
1. Neurotrophins Play Important Role in Cortical Injury Recovery
Brief depolarization of primary septo-hippocampal cell cultures can produce significant losses of neurofilament proteins. Studies have indicated that brain-derived neurotrophic factor (BDNF) increases neurofilaments in hippocampal cell cultures (Yip et al., 1993) and increases survival of cortical neurons (Ghosh et al., 1994).
Several articles have reported attempted treatments for treating traumatic brain injury. Exogenous supplementation of NGF has been reported to spare neurons from death and degeneration following injury (Hafti, 1986; Kromer, 1987; Montero and Hafti, 1988; Williams et al., 1986) and to increase choline acetyl transferase (CHAT) activity (Rylett et al., 1993; Williams and Rylett, 1990).
NGF prevents degeneration of septal cholinergic neurons following fimbria lesions or transfection (see e.g., Morse et al., 1993). NGF and BDNF increase survival of septal cholinergic neurons in vitro (see e.g., Alderson et al., 1990) and increase choline acetyl transferase (ChAT) activity both in vitro (see e.g., Alderson et al., 1990) and in intact animals (see e.g., Rylett et al., 1993).
Observations indicate that TBI can result in disturbances of cholinergic neurotransmission associated with impaired release of acetylcholine in the hippocampus (Dixon et al., 1993). In some cases, this disruption may cause nerve death, which may ultimately lead to brain dysfunction. However, the mechanism of cholinergic disturbance is not well-understood, and therapies addressing this abnormality in vivo have not been reported.
2. NGF and BDNF Expression in the Hippocampus
NGF and BDNF are members of the neurotrophin gene family, as are neurotrophin-3, and neurotrophins-4/5 (NT-3, NT-4/5). All are expressed in brain (for review, see Lindsay, 1993). During development in the adult rat brain BDNF and NGF mRNA are particularly abundant in the hippocampus (Maisonpierre et al., 1990b; Ernfors et al., 1990), a region which is preferentially vulnerable to TBI (Hayes et al., 1992; Olenik et al., 1988).
3. BDNF and NGF Blunt Free Radical Damage
Formation of free radicals and subsequent lipid peroxidation may contribute to TBI (Chan, 1992; Hall et al., 1992). Giving NGF to adult rats protects their sympathetic ganglia from 6-hydroxydopamine toxicity, a free radical generator (Johnnson, 1980). Furthermore, in adult rats, long-term NGF administration increases the activity of antioxidant enzymes in cortex (Nistico et al., 1992). Protection by NGF in cultures of PC-12 cells is associated with specific increases in catalase activity and glutathione levels due to stimulation of glutathione redox reactions and synthesis (see e.g., Sampath et al., 1993).
4. BDNF and NGF Initiate Cytoskeleton Repair
NGF has been shown to initiate and maintain neurite outgrowth in rat pheochromocytoma (PC12) cells (Greene and Tischler, 1982), and is associated with increased levels of .beta.-tubulin (see e.g., Teng and Greene, 1993). BDNF has been shown to increase in vitro levels of neurofilament in hippocampal cell cultures (Yip et al., 1993). However, the role of these two growth factors in cytoskeletal stabilization in vivo had not been determined.
5. BDNF and NGF Are Involved in Neurodegenerative Disease
TBI is a risk factor for Alzheimer's disease (Mortimer et al., 1991), and in some cases, is also associated with diffuse deposition of .beta./A.sub.4 protein (Roberts et al., 1991; Clinton et al., 1991), the amyloidogenic protein of Alzheimer's disease. Furthermore, in Alzheimer's disease in situ hybridization reveals significant decreases in BDNF mRNA (Phillips et al., 1991). In aged rats, NGF increases high affinity choline transport (Williams et al., 1990), stimulates Ach release (Rylett et al., 1993) and also improves the performance of age-impaired rats in spatial memory tasks (Fischer et al., 1987).
6. BDNF and NGF Expression After CNS Injury is Only Transient
Although various injuries to the CNS up-regulate the production of neurotrophins and increase BDNF and NGF mRNA in the hippocampus (Yang et al., 1993), in most injuries, production of neurotrophins is not sustained long enough to promote recovery. In TBI, prominent up-regulation of BDNF mRNA occurs in the hippocampus, but only transiently, from 1 h to 6 h after injury (Yang et al., 1993), and therefore levels of neurotrophins are not present in substantial levels to effect cytoskeletal (e.g., neurofilament) rescue.
7. Providing Neurotrophins to CNS Cells is Desirable Following TBI
Many different approaches have been contemplated to deliver exogenous neurotrophins to the nervous system of mammals (Hafti, 1986; Kromer, 1987; Montero and Hafti, 1988; Williams et al., 1986), although significant limitations imposed by protein degradation and by the blood-brain barrier have restricted the clinical utility of these approaches (Barinega, 1994).
Unfortunately, even though exogenous growth factors can be delivered within the brain by means of mini-osmotic pumps that release small amounts of neurotrophin into the ventricular cavity or directly into the parenchyma (Hafti, 1986; Williams et al., 1986; Kromer, 1987; Montero and Hafti, 1988), pumps must be mechanically regulated, and often can fail. This method is particularly expensive and inconvenient, since the stored growth factors in the pump reservoir diminish in activity over time, and fresh amounts of the neurotrophin must be constantly added.
Many drawbacks are also associated with these types of treatment protocols, not the least of which is the expensive and time-consuming purification of the recombinant proteins from their host cells (Rylett et al., 1993; Morse et al., 1993). Also, polypeptides, once administered to an animal are more unstable than is generally desired for a therapeutic agent, and they are susceptible to proteolytic attack. Furthermore, the administration of recombinant proteins can initiate various inhibitive or otherwise harmful immune responses.
8. Focal Delivery of Neurotrophins
Investigators studying central nervous system injury have long recognized that changes in specific proteins may be important determinants of pathological responses to injury and recovery of function of the injured brain. Gene transfer has emerged as a potential way to introduce neurotrophins into CNS cells and tissues (Friedmann and Ginnah, 1993). Methods currently used to introduce genes into localized regions of the nervous system through stereotactic injection include retroviral vectors, herpes virus vectors, adenoviral vectors and grafted cells. Grafted cells have been most extensively examined for growth factor production.
Studies grafting cells in brain capable of producing growth factors have employed mouse sarcoma cells, male mouse submaxillary gland cells (Levi-Montalcini and Cohen, 1960; Caramia et al., 1962) and sciatic nerve cells (Richardson and Ebendal, 1982) or alternatively, in cell culture followed by implantation within the brain (Gage et al., 1987). Despite limited success with several different cell types, including primary fibroblasts (Kawaja et al., 1992), astrocytes and immortalized cell lines (Rosenberg et al., 1988; Wolf et al., 1988) grafts remain only a temporary solution, since most will eventually be rejected.
Although retroviral vectors are considered the most efficient vectors for stable gene transfer into mitotic mammalian cells and have been widely used in CNS cancer gene therapy (Yamada et al., 1992; Ram et al., 1993; Culver et al., 1992), they are unsuitable for post-mitotic CNS cells, since with the exception of some lentiviruses, retroviruses can integrate only into chromosomes of dividing cells. Another significant limitation of retroviral vectors is their maximum insert capacity of 5-7 kb (Gelinas and Teman, 1986).
Two methods using viral vectors have been described: 1) Herpes Simplex virus, and 2) adenovirus-mediated delivery systems. Unfortunately, however, neither are suitable for treatment of TBI. Although Herpes virus is able to infect post-mitotic cells and can be taken up anywhere along the cell surface (Breakefield and DeLuca, 1991; Lycke et al., 1988), its toxicity to nervous cells (Johnson et al., 1992) and its disruption of normal neuronal architecture (Huang et al., 1992) make it unsuitable for treatment of TBI and CNS cells.
Replication-deficient adenoviral vectors have also been used to infect rat CNS cells in vitro and in vivo, since these DNA viruses are able to infect post-mitotic cells (le Gal La Salle et al., 1993), but unfortunately, they do not integrate efficiently into the nuclear DNA of the recipient cells, gene expression occurs only transiently, often in an unpredictable manner (Graham and Prevec, 1991; Horwitz, 1990). Further limiting these methods in the treatment of TBI is their limited transfection into neural cells, and the pathogenicity and limited duration of gene expression of these vectors.
Several groups have investigated the possibility of using liposomes as a means of mediating delivery of genetic information into nervous system cells in vitro, but unfortunately these methods were disappointing. For example, using the E. coli .beta.-Gal reporter gene in liposome-mediated transfection of low density cultures of rat hippocampal neurons, the transfection efficiency was less than 1% of the whole cell population, and the small fraction of transfected cells was mostly neuronal (Drazba and Ralston, 1993).
Likewise, an ex vivo method employing cationic liposomes to transfect primary rodent neuronal cell cultures with a gene encoding .beta.-Gal (Ono et al., 1990) showed only limited success, when the cell types which incorporated and expressed the injected cDNA were not delineated.
9. Deficiencies in the Prior Art
A method of treating a variety of central nervous system pathologies through manipulations of specific trophic and/or toxic proteins would have important therapeutic potential. Increasing expression of trophic factors which have been shown to enhance recovery of function following trauma to the brain and spinal cord would be particularly desirable. More than 500,000 patients annually are hospitalized for traumatic brain injury. More than 10,000 patients are treated for spinal cord injury and 750,000 patients for stroke. Neurodegenerative diseases, organically-based psychological disorders and chronic pain are all widely recognized major health problems in the United States. The presence of the blood-brain barrier significantly confounds the choice of routes for administration of neurotrophins.
It is clear, therefore, that a new method capable of promoting nervous system cell repair and regeneration in vivo would represent a significant scientific and medical advance with immediate benefits to a large number of patients. A method readily adaptable for use with a variety of growth factors and other genes would be particularly advantageous. Rescue of neurofilament loss due to TBI by increasing the availability of neurotrophins both ex vivo and in vivo would represent a significant improvement in the treatment of central nervous system injury.